Ultra high throughput DNA sequencing system based on two-dimensional monolith mul

基于二维整体式测序仪的超高通量DNA测序系统

• 批准号: 6960958
• 负责人: VERA B. GORFINKEL
• 金额: $ 75万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6960958
• 关键词: biomedical automation; biotechnology; capillary electrophoresis; high throughput technology; nucleic acid sequence; technology /technique development

DESCRIPTION (provided by applicant): The main goal of the present project is a development, implementation and testing of a highly efficient method and an instrument for DNA sequencing capable of both automated re-sequencing and de-novo sequencing of mammalian size genomes with the mouse draft quality {Nature 420:520, 2002} at 100-fold reduced cost. The proposed system will be based on highly parallel CE separation and detection of fluorescently labeled dideoxynucleotide-terminated DNA extension product generated by gel matrix-immobilized colonies of single template molecules (polonies) in two dimensional monolith multi-capillary arrays (2D-MMCA). The cost reduction will come from both using nanoliter volume reactions and employing 2D-MMCAs which increase the throughput of the CE separation and detection by at least two orders of magnitude compared to commonly used high-throughput DNA machines. We will perform the following: Development of the technology platform, building of a pilot 55x55 lane automated DNA sequencer capable of sequencing 1,000 bp/second with 450bp Q20 read length at cost of $0.007/kbp, and demonstration of the production scale re-sequencing and automated generation of a genome sequence of the quality of the mouse draft genome at less than $100,000 per mammalian size genome Further development of the proposed system and introducing the technology and the instrumentation changes enabling de-novo sequencing: Optimization of polonie's cultivation technology and obtaining an efficient amplification and cycle sequencing of 3-5kb DNA fragments; Building a pilot 100x300 lane DNA sequencer capable of automated generation of raw sequencing data at 7500 bp/second with 800 bp Q20 read length at cost of $0.002/kbp. Demonstration of the system's potential for de-novo sequencing of human size genome at 10 fold coverage for $100,000 - $200,000.

描述（由申请人提供）：本项目的主要目标是开发、实施和测试用于DNA测序的高效方法和仪器，其能够以100倍降低的成本对具有小鼠草稿质量的哺乳动物大小的基因组进行自动再测序和从头测序{Nature 420：520，2002}。所提出的系统将基于高度平行的CE分离和检测的荧光标记的双脱氧核苷酸终止的DNA延伸产物所产生的凝胶基质固定的单模板分子（polonies）的菌落在二维整体多毛细管阵列（2D-MMCA）。成本降低将来自使用纳升体积反应和采用2D-MMCAs，与常用的高通量DNA机器相比，这将CE分离和检测的通量提高了至少两个数量级。 我们将执行以下操作：技术平台的开发，能够以0.007美元/kbp的成本以1,000 bp/秒的速度测序450 bp Q20读长的55x 55泳道自动DNA测序仪的建立，以及以低于100美元的成本演示生产规模的重新测序和自动生成小鼠基因组草图质量的基因组序列，进一步开发所提出的系统，并引入技术和仪器变化，使从头测序成为可能：优化polonie的培养技术，并获得3- 5 kb DNA片段的有效扩增和循环测序;构建中试100 × 300泳道DNA测序仪，其能够以7500 bp/秒的速度自动生成原始测序数据，具有800 bp Q20读取长度，成本为0.002美元/kbp。证明该系统在10倍覆盖率下对人类大小的基因组进行从头测序的潜力，费用为10万至20万美元。